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REPUBLIC OF NAMIBIA

Ministry of Agriculture Water and Forestry Directorate of Veterinary Services

April 2017

Mycobacterium bovis Surveillance Protocol

Compiled by:

Division of Epidemiology, Import & Export, Traceability, Medicine Control and Advisory Service

Private Bag 12022

Windhoek / Namibia

Tel: +264-61-2087542

Fax: 264-61-2087779

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Table of Contents 1. INTRODUCTION ......................................................................................................................... 3

2. SURVEILLANCE STRATEGY ......................................................................................................... 3

2.1 Surveillance at slaughter facilities....................................................................................... 4

2.1.1 Clinical Case definition ................................................................................................ 4

2.1.2 Post mortem Case Definition ...................................................................................... 4

2.1.3 Differential Diagnosis.................................................................................................. 4

2.2 Testing of animals during import/export ............................................................................ 5

2.3 Investigation of reported suspect cases .............................................................................. 5

3. SAMPLING FOR MYCOBACTERIUM BOVIS AT POST-MORTEM .................................................. 5

3.1 Sampling for laboratory investigation ................................................................................. 6

4. LABORATORY DIAGNOSIS ......................................................................................................... 6

4.1 Polymerase Chain Reaction (PCR) ....................................................................................... 6

4.2 Microscopic examination ................................................................................................... 7

4.3 Culture ............................................................................................................................... 7

5. TRACE BACK INVESTIGATION .................................................................................................... 7

6. TRAINING .................................................................................................................................. 7

7. REPORTING AND DISSEMINATION ............................................................................................ 8

8. ANNEXES ................................................................................................................................... 9

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1. INTRODUCTION

Bovine tuberculosis is a chronic bacterial disease of animals and humans caused by

Mycobacterium bovis. In a large number of countries bovine tuberculosis is a major infectious

disease among cattle, other domesticated animals, and certain wildlife populations. The

disease can cause production losses when poorly controlled and is classified by the World

Organisation for Animal Health (OIE) as a disease of socio-economic, public health and trade

importance. The public health risk has been alleviated in many countries through

pasteurisation of milk.

Bovine Tuberculosis, caused by the bacterium Mycobacterium bovis, is listed as a notifiable

disease in Notice No180 of Government Gazette No. 5239 of 12 July 2013. Currently, Namibia

regards itself as free from Bovine Tuberculosis. The last case diagnosed in a live bovine was

in 1994 and in a slaughtered bovine in 1995. No cases have been detected since then.

Aerosol exposure to M. bovis is considered to be the most frequent route of infection of

cattle, but infection by ingestion of contaminated material also occurs. After infection,

nonvascular nodular granulomas known as tubercles may develop. Characteristic tuberculous

lesions occur most frequently in the lungs and the retropharyngeal, bronchial and mediastinal

lymph nodes. Lesions can also be found in the mesenteric lymph nodes, liver, spleen, on

serous membranes, and in other organs.

Bovine tuberculosis infection in cattle is usually diagnosed in the live animal on the basis of

delayed hypersensitivity reactions using the single or comparative intradermal tests. Infection

is often subclinical; when present, clinical signs are not specifically distinctive and can include

weakness, anorexia, emaciation, dyspnoea, enlargement of lymph nodes, and cough,

particularly with advanced tuberculosis. After death, infection is diagnosed by necropsy and

histopathological and bacteriological techniques. Rapid nucleic acid methodologies, such as

the polymerase chain reaction (PCR), may also be used although these are demanding

techniques and should only be used when appropriately validated. Traditional mycobacterial

culture remains the gold standard method for routine confirmation of infection.

2. SURVEILLANCE STRATEGY

Mycobacterium bovis surveillance strategy is implemented through active and passive

surveillance. Active surveillance involve examination of carcasses at slaughter facilities and

testing of animals before export and after import using the intradermal tuberculin. The

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passive surveillance involves investigation of reported suspect clinical cases through

intradermal testing or post mortem examination of organs.

2.1 Surveillance at slaughter facilities

There are seven export approved abattoirs and local authority slaughter facilities distributed

throughout the country. The Directorate of Veterinary Services (DVS) provides oversight at

the export approved abattoirs while the local authority slaughter facilities are under the

supervision of Local Authorities under the Ministry of Regional and Local Government,

Housing and Rural Development and the Ministry of Health and Social Services.

Abattoir surveillance through ante and post mortem inspection is ongoing. However, in order

to collect more credible data, a sampling protocol has been developed to enable diagnosis of

all lesions that resemble bovine tuberculosis.

Meat inspection is conducted by qualified and registered meat inspectors and is done

according to the Meat Safety Act, 2000 (Act No. 40 of 2000), Red Meat Regulations (Act No.

40 of 2000) and the guidelines of the South African Meat Inspectors Manual for Red Meat. At

the export approved abattoirs meat inspection is done by Veterinary Hygiene Inspector

Assistants and Veterinary Hygiene Inspectors under the supervision of State Veterinarians. At

non-export slaughter facilities meat inspection is done by meat inspectors with a minimum of

a Diploma in Environmental Sciences.

2.1.1 Clinical Case definition

Bovine tuberculosis can be suspected when some of the following signs are observed during

ante-mortem inspection:

Weakness

Emaciation

Dyspnoea

Coughing

Enlargement of lymph nodes especially those in the neck region

2.1.2 Post mortem Case Definition

Bovine tuberculosis can be suspected when some of the following signs are observed during

post-mortem inspection

Nodular granulomas in organs including lungs, intestines, liver spleen, pleura and

peritoneum

Nodular granulomas in lymph nodes especially of head and neck

Small nodular granulomas on thoracic wall (miliary)

2.1.3 Differential Diagnosis

If the following diseases and conditions and conditions are suspected samples must be

collected to rule out bovine tuberculosis:

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Contagious bovine pleuropneumonia (with sequestra present)

Actinobacillosis

Lung abscessation

Bovine farcy (Mycobacterium farcinogenes)

Bacterial or viral bronchopneumonia

Echinococcal hydatid cysts (calcified)

2.2 Testing of animals during import/export

All cattle destined for export to neighbouring countries are subjected to the single

intradermal tuberculin test as per the Protocol on Bovine Tuberculosis Testing. Similarly all

cattle imported into the country are subjected to the single intradermal tuberculin test. Data

from such testing of cattle is compiled to give a summary of number of cattle and number of

herds tested and the results of such testing.

2.3 Investigation of reported suspect cases

State Veterinarians in the field are responsible for investigating suspected outbreaks of

disease. Any mycobacterium tuberculosis suspect whether clinical or post mortem is

investigated through appropriate intradermal tuberculin test or sample collection for

bacterial culture or PCR. The handling of suspect cases at post-mortem will be as for

investigation at slaughter facilities.

3. SAMPLING FOR MYCOBACTERIUM BOVIS AT POST-MORTEM

At post-mortem, tubercles are most frequently seen in bronchial, mediastinal,

retropharyngeal and portal lymph nodes and may be the only tissue affected. In addition, the

lung, liver, spleen and the surfaces of body cavities are commonly affected. Early nodular

pulmonary lesions can often be detected by palpation. The lesions are usually non-

odoriferous. Other anatomical sites can be infected and should be examined.

Figure 1. Typical Mycobacterium bovis granuloma.

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A tuberculous granuloma has a yellowish appearance

It has a caseous, caseo-calcereous consistency

Its appearance may be purulent

The caseous centre is usually dry, firm and covered with a fibrous connective capsule

Lesion size range from small to involvement of greater part of organ, hence serial

sectioning of tissues required to detect small lesions

3.1 Sampling for laboratory investigation

1. Lesions suggestive of bovine tuberculosis must be sampled and submitted for

diagnosis to the Central Veterinary Laboratory.

2. Collection instruments and containers should be clean and preferably sterile (use of

instruments and containers that are contaminated by environmental mycobacteria

may result in the failure to identify M. bovis infection due to the rapid growth of the

environmental mycobacteria); where feasible, single-use plastic, disposable

containers, 50 ml in capacity, may be used for a variety of specimen types.

3. The container must be CLEARLY labelled containing the following minimum

information: species, organ/tissue sampled, animal identification number.

4. A QUA from 99 must be completed to accompany the samples.

5. Specimens must be cushioned and sealed to prevent leakage, and properly packaged

to withstand breakage or crushing in transit.

6. Delivery of samples to CVL must be prompt. Prompt delivery of specimens to the

laboratory greatly enhances the chances of cultural recovery of M. bovis. If delays in

delivery are anticipated, specimens should be refrigerated or frozen to retard the

growth of contaminants and to preserve the mycobacteria.

4. LABORATORY DIAGNOSIS

The Central Veterinary Laboratory (CVL) is responsible for laboratory confirmation for

Mycobacterium bovis and forwarding samples to other laboratories for confirmation.

Precautions should be taken to prevent infection of laboratory personnel. All procedures

involving preparation of the tissue samples should be performed in a biological safety cabinet.

4.1 Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) has been successfully applied to detect members of the M.

tuberculosis complex and is especially useful for the direct detection of M. bovis in bovine

tissue samples. Any positive result should be confirmed by culture.

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4.2 Microscopic examination

Mycobacterium bovis can be demonstrated microscopically on direct smears from clinical

samples and on prepared tissue materials.

The acid fastness of M. bovis is normally demonstrated with the classic Ziehl - Nielsen stain,

but a fluorescent acid-fast stain may also be used. Immunoperoxidase techniques may also

give satisfactory results.

The presumptive diagnosis of mycobacteriosis can be made if the tissue has characteristic

histological lesions (caseous necrosis, mineralisation, epithelioid cells, multinucleated giant

cells and macrophages).

4.3 Culture

Cultures are incubated for a minimum of 8 weeks (and preferably for 10–12 weeks) at 37°C

with or without CO2. The media should be in tightly closed tubes to avoid desiccation. Slopes

are examined for macroscopic growth at intervals during the incubation period.

When growth is visible, smears are prepared and stained by the Ziehl–Nielsen technique.

Growth of M. bovis generally occurs within 3–6 weeks of incubation depending on the media

used. Characteristic growth patterns and colonial morphology can provide a presumptive

diagnosis of M. bovis; however every isolate needs to be confirmed. It is necessary to

distinguish M. bovis from the other members of the ‘tuberculosis complex’.

Currently the CVL is using PCR for M bovis diagnosis while culture and biochemical tests are

referred to South Africa.

5. TRACE BACK INVESTIGATION

Any positive result from slaughter facility must be reported to Animal Disease Control Division

to enable trace back to the farm/ village of origin by the local State Veterinarian.

6. TRAINING

All staff responsible for implementing the M. Bovis surveillance in DVS will be subjected to

training in intradermal tuberculin testing, gross pathological diagnosis and sample collection

and preservation. The gross pathological diagnosis training will also cover differential

diagnosis for M bovis aforementioned. The training for CBPP gross pathological diagnosis will

be very important for the Northern Communal Areas where CBPP freedom is one of the

objectives. Staff from Ministry of Health and Social Services and Local Authorities responsible

for meat inspection will also be trained in gross pathological diagnosis and sample collection

and preservation.

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7. REPORTING AND DISSEMINATION

All surveillance activities for Mycobacteria bovis are to be reported in Monthly Reports for

inclusion in the National Summary Reports for communication to stakeholders.

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8. ANNEXES

Annex 1. Differential Diagnosis for M bovis

1. Contagious bovine pleuropneumonia

CBPP sequestra in lung tissue

Grey hepatisation of lung due to CBPP

Marbling appearance of lung due to CBPP

2. Actinobacillosis

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Lung lesions due to actinobacillosis

3. Lung abscessation

Multiple abscesses in a lung

4. Bovine farcy (Mycobacterium farcinogenes)

Characterised by pneumonia, abscessation, mastitis, cutaneous and subcutaneous lesions

5. Bacterial or viral bronchopneumonia

Bronchopneumonia in cattle

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6. Echinococcal hydatid cysts (calcified)

Lung hydatid cyst with well defined membrane